Methods of analyzing biological samples using a fluidic cartridge

ABSTRACT

A method for analyzing biological samples is disclosed herein. In an embodiment, the method includes receiving a fluid sample into a cartridge device, which comprises: a fluidic chamber; at least one microfluidic channel in fluid communication with the fluidic chamber; and a venting port configured to apply a pneumatic force to the fluidic chamber; and inserting the cartridge device into a reader device to perform measurements, wherein the cartridge device is positioned in a vertical or tilted position such that at least a portion of the fluid sample inside the fluidic chamber is pulled by gravity in a direction away from the venting port or towards the bottom of the fluidic chamber.

PRIORITY CLAIM

This application is a continuation of U.S. application Ser. No.15/176,729, filed Jun. 8, 2016, entitled “Fluidic Units and Cartridgesfor Multi-Analyte Analysis”, which claims priority to U.S. ProvisionalPatent Application No. 62/174,776, entitled “Fluidic Units andCartridges for Multi-Analyte Analysis”, filed Jun. 12, 2015, the entirecontents of which are hereby incorporated by reference and relied upon.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to methods and apparatuses formulti-analyte analyses, and more specifically to fluidic units andcartridges for multi-analyte analyses.

BACKGROUND OF THE DISCLOSURE

In the field of in-vitro diagnostics, analyses are often made onbiological samples, such as body fluids (e.g. blood, urine, saliva,cerebrospinal fluid, etc.), cell suspension (e.g. tissue cellssuspension in buffer), and other fluid samples. For certain scenarios,such as point-of-care testing, it is desirable to perform the aboveanalysis in the format of a cartridge device. It is also desirable thatthe cartridge is disposable after each use to avoid cross-contamination.Microfluidic technologies can be used to build these cartridge devices,due to the merits of a small sample volume and a small cartridge size.However, cartridge devices that can be used to measure multiplebiological markers are still lacking.

SUMMARY OF THE DISCLOSURE

In one aspect, the present disclosure is directed to a design of afluidic unit to construct cartridges for testing biological samples. Thefluidic unit comprises a chamber, a venting port and at least onemicrofluidic channel that accesses the chamber and has a passive valve.The operation of this unit depends on gravity or another force as areplacement for gravity, such as a centrifugal force, to keep fluid inposition. In addition, it uses another force such as pneumatic pressureto transfer fluid. The design of this fluidic unit has an intrinsic“self-stop” mechanism, which is configured to overcome the challenge ofaccurate volume transfer in pneumatically actuated cartridges. Thisfluidic unit can be modified to achieve various fluidic functions, suchas mixing samples, removing bubbles, transferring a fixed-volume,relaying fluid flow, etc. With a plurality of these fluidic units, moresophisticated fluidic functions can be achieved, such as serialdilution, replacing T-junctions in microfluidic, etc. The fluidic unitcan also be implemented by a structure including a plurality of layers.In an embodiment, the fluidic unit can be implemented as a two-layerstructure. In another embodiment, the fluidic unit can be implemented asa three-layer structure.

The present disclosure also explains how to design cartridges with thefluidic unit to test various biological samples. The cartridges can havea plurality of design units and optionally other fluidic components suchas fluidic conducts, valves and pumps, etc. The cartridge devices can beused for tests such as Complete Blood Count, Flow Cytometer Analysis,Blood Chemistry, Blood Gas, Immunoassay, Nucleic Acid purification, andMolecular Diagnostics, etc. The present disclosure is optimal forintegrating multiple of the above tests into one cartridge.

For embodiments of cartridges that depend on gravity, it is desirable tomaintain a vertical position. In these cartridges, the accuracy oftransferring a fixed volume of fluid is sensitive to tilting away fromthe vertical position. The present disclosure introduces methodologiesto reduce or eliminate the effect of tilting.

The cartridges are inserted into a reader to read out measurementsignals. The present disclosure is also directed to multiple readerdesigns to work with cartridges of the fluidic unit. One reader deviceaccepts only one cartridge at a time. Another reader device cansimultaneously accept multiple cartridges, wherein the cartridges arerun in serial, in parallel or in streamline to increase test throughput.

In a general example embodiment, a fluidic device includes a fluidicchamber, at least one microfluidic channel in fluid communication withthe fluidic chamber, a venting port configured to apply a pneumaticforce to the fluidic chamber, at least one passive valve located withinthe at least one microfluidic channel and configured to allow or stopfluid flow through the at least one microfluidic channel based on apressure difference, and a controller configured to control thepneumatic force applied to the fluidic chamber via the venting port.

In another embodiment, the fluidic chamber is located within adisposable cartridge configured to be held in a vertical position by ahousing of the fluidic device.

In another embodiment, the venting port is located at a top portion ofthe fluidic chamber when the fluidic chamber is held in the verticalposition.

In another embodiment, the at least one microfluidic channel is locateda height below the venting port when the fluidic chamber is held in thevertical position.

In another embodiment, the at least one microfluidic channel includes afirst microfluidic channel and a second microfluidic channel, the firstmicrofluidic channel located a height above the second microfluidicchannel with the fluidic chamber is held in the vertical position.

In another embodiment, the at least one passive valve includes at leastone of: (i) a hydrophobic patch; (ii) a hydrophilic patch; (iii) asudden diameter enlargement of a hydrophobic channel; and (iv) a suddendiameter shrink of a hydrophobic channel.

In another embodiment, the controller is configured to control thepneumatic force applied to the fluidic chamber via the venting portbased on (i) a pressure (P₀) associated with the venting port, and (ii)a pressure (P₁) associated with the at least one microfluidic channel.The pressure difference (P₀−P₁) provides the pneumatic force to drivefluid and air.

In another embodiment, the controller is configured to store fluid inthe fluidic chamber by controlling the pneumatic force applied to thefluidic chamber via the venting port according to the followingequations: −ΔP_(in)−ρgh≤P₀−P₁≤ΔP_(out)−ρgh, if h≥0; and P₁−P₀≤ΔP_(in),if h<0, wherein (i) ΔP_(in) is a first threshold pressure associatedwith a first direction of fluid entering the fluidic chamber, (ii)ΔP_(out) is a second threshold pressure associated with a seconddirection of fluid leaving the fluidic chamber, and (iii) ρgh is thehydraulic pressure of the fluid that is caused by the gravity or areplacement for gravity such as centrifugal force.

In another embodiment, the controller is configured to transfer fluidinto the fluidic chamber by controlling the pneumatic force applied tothe fluidic chamber via the venting port according to the followingequations: P₁−P₀>ΔP_(in)+ρgh, if h≥0; and P₁−P₀>ΔP_(in), if h<0, wherein(i) ΔP_(in) is a threshold pressure associated with a direction of fluidentering the fluidic chamber, and (ii) ρgh is the hydraulic pressure ofthe fluid.

In another embodiment, the controller is configured to transfer fluidout of the fluidic chamber by controlling the pneumatic force applied tothe fluidic chamber via the venting port according to the followingequation: P₀−P₁>ΔP_(out)−ρgh, wherein (i) ΔP_(out) is a thresholdpressure associated with a direction of fluid leaving the fluidicchamber, and (ii) ρgh is the hydraulic pressure of the fluid.

In another embodiment, the fluidic chamber includes a filter membranewith a pore size smaller than known particles in the fluid.

In another embodiment, the device includes a plurality of fluidicchambers, and wherein the controller controls the pneumatic pressureapplied to respective venting ports of the plurality of fluidic chambersindependently of each other.

In another embodiment, the plurality of fluidic chambers includes afirst fluidic chamber and a second fluidic chamber, the first fluidicchamber and the second fluidic chamber in fluid communication via onlyone microchannel.

In another general example embodiment, a fluid testing system includes adevice including a pneumatic source and a controller configured tocontrol the pneumatic source, and a fluidic cartridge configured to beinserted into the device, the fluidic cartridge including an inlet portconfigured to receive a fluid sample, a sample retaining chamberconfigured to receive the fluid sample from the inlet port, a firstfluidic chamber configure to store or receive a reagent, the firstfluidic chamber in fluid communication with the sample retainingchamber, and a second fluidic structure in fluid communication with thesample retaining chamber.

In another embodiment, the controller is configured to mix the fluidsample with the reagent in the second fluidic structure by activatingthe pneumatic source to cause the reagent from the first fluidic chamberto flush the fluid sample into second fluidic structure.

In another embodiment, the second fluidic structure includes a sensingstructure, and wherein the controller is configured to push the fluidsample first and the reagent second through the sensing structure.

In another embodiment, the second fluidic chamber includes a filtermembrane with a pore size smaller than target cells in the fluid sample.

In another embodiment, the sample retaining chamber is positioned andarranged to draw the fluid sample through the inlet port by capillaryforce.

In another general example embodiment, a fluidic device includes afluidic chamber, at least one microfluidic channel in fluidcommunication with the fluidic chamber, a tilt sensor configured tosense a tilt angle of the fluidic chamber, and a controller configuredto determine a volume of fluid to be pumped into or out of the fluidicchamber via the at least one microfluidic channel based on the tiltangle sensed by the tilt sensor.

In another embodiment, the device includes a venting port configured toapply a pneumatic force to the fluidic chamber, and wherein thecontroller is configured to control the pneumatic force applied to thefluidic chamber via the venting port to expel the volume of fluid fromthe fluidic chamber.

In another embodiment, the controller is configured to determine thevolume of fluid based on a shape of the fluidic chamber and the tiltangle sensed by the tilt sensor.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure will now be explained in furtherdetail by way of example only with reference to the accompanyingfigures, in which:

FIG. 1 shows the design of an example embodiment of a fluidic unitaccording to the present disclosure;

FIGS. 2A to 2D show example embodiments of passive valves that can beused with the fluidic unit of FIG. 1;

FIG. 3 shows the design of an example embodiment of a fluidic unitaccording to the present disclosure;

FIGS. 4A to 4I show an example embodiment of fluid transfer with anexample embodiment of a fluidic unit according to the presentdisclosure;

FIGS. 5A and 5B show an example embodiment of fluid transfer with anexample embodiment of a fluidic unit according to the presentdisclosure;

FIGS. 6A and 6B show an example embodiment of fluid transfer with anexample embodiment of a fluidic unit according to the presentdisclosure;

FIGS. 7A and 7B show an example embodiment of fluid transfer with anexample embodiment of a fluidic unit according to the presentdisclosure;

FIGS. 8A to 8J shows the designs of example embodiments of fluidic unitsaccording to the present disclosure;

FIGS. 9A and 9B show an example embodiment of fluid transfer with anexample embodiment of a fluidic unit according to the presentdisclosure;

FIGS. 10A to 10D show an example embodiment of fluid transfer with anexample embodiment of a fluidic unit according to the presentdisclosure;

FIGS. 11A to 11C show an example embodiment of fluid transfer with anexample embodiment of a fluidic unit according to the presentdisclosure;

FIGS. 12A to 12C shows the designs of example embodiments of fluidicunits according to the present disclosure;

FIGS. 13A to 13E show an example embodiment of fluid transfer with anexample embodiment of a fluidic unit according to the presentdisclosure;

FIGS. 14A to 14F show example embodiments of fluidic units with reagentsaccording to the present disclosure;

FIGS. 15A to 15C show example embodiments fluidic units with filtermembranes according to the present disclosure;

FIG. 16 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIGS. 17A and 17B show example embodiments of a fluidic circuitsaccording to the present disclosure;

FIG. 18 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIGS. 19A to 19D show an example embodiment of fluid transfer with anexample embodiment of a fluidic circuit according to the presentdisclosure;

FIG. 20A to 20C show example embodiments of fluidic circuits accordingto the present disclosure;

FIGS. 21A to 21C show example embodiments of fluidic units with aplurality of layers according to the present disclosure;

FIGS. 22A to 22C show example embodiments of fluidic units with aplurality of layers according to the present disclosure;

FIG. 23A shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 23B shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 24 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 25 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 26 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 27 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 28 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 29 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 30 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 31 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 32 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIG. 33 shows an example embodiment of a fluidic circuit according tothe present disclosure;

FIGS. 34A to 34G show how embodiments of the present disclosure accountfor tilting of a fluidic cartridge.

DETAILED DESCRIPTION

FIG. 1 shows the design of a fluidic unit 1001, which comprises achamber 1002, a venting port 1003 and a microfluidic channel 1004 thatis in fluid communication with and accesses the chamber and includes apassive valve 1005. The fluidic unit can be used to handle differentfluids, such as liquid, liquid containing bubbles, or liquid containingparticles. The fluids can be body fluids (e.g. blood, urine, saliva,etc.), reagent solutions, beads suspended in buffer, etc.

The fluidic chamber 1002 provides an enclosed space to receive and storefluid. Fluidic chamber 1002 is designed so that the fluid can sink or bepulled down to the bottom of the chamber and bubbles can float up to thetop, either by gravity or other forces such as a pneumatic orcentrifugal force. One way to achieve this property is to have fluidicchamber 1002 dimensioned large enough so that the gravity is moredominant than the surface tension of the fluid. It can also be achievedin other ways, for example, by applying a centrifugal force moredominant than the surface tension. A preferred dimension of fluidicchamber 1002 is 0.1 mm to 50 mm in width, 0.1 mm to 50 mm in depth, and0.1 mm to 100 mm in height. Fluidic chamber 1002 could be any shape, forexample, cuboid, cylindrical, spherical or other shapes of containersknown to persons skilled in the art, with dimensions in the aboveranges.

The venting port 1003 is configured to apply pneumatic pressure tofluidic chamber 1002. Venting port 1003 should be positioned above thefluid when the device is in use. Venting port 1003 can be below thefluid when the device is in storage or other states of nonuse. It can beof any size and of any surface property. Preferably, venting port 1003is a micro-sized channel with a hydrophobic surface, where surfacetension is more dominant than gravity. The pneumatic pressure applied tothe venting port 1003 can be of atmosphere pressure, a pressure higherthan the atmosphere, or a vacuum lower than the atmosphere. When theventing port 1003 is connected to an atmosphere pressure, it can act asa pressure buffer to keep the pressure inside the chamber constantlyequalized to atmosphere.

The microfluidic channel 1004 is in fluid communication with andaccesses the enclosed space of fluidic chamber 1002. Preferably,microfluidic channel 1004 is a micro-sized channel where surface tensionis more dominant than gravity. In an embodiment, microfluidic channel1004 has a cross section of 0.1 um to 5 mm in width and 0.1 um to 5 mmin depth. The cross section can be in shape of a rectangle, a trapezoid,a cylinder or any other shapes known to persons skilled in the art.Additionally, microfluidic channel 1004 includes a passive valve 1005,which stops fluid flow if the pressure difference across the fluidmeniscus is below a designated threshold ΔP. The positioning of valve1005 is preferably close to the chamber, so that the fluid volumebetween the valve and the chamber is negligible in comparison to thefluid volume being manipulated. In example embodiment, passive valve1005 can be a hydrophobic patch (FIG. 2A), a hydrophilic patch (FIG.2B), a sudden diameter enlargement of a hydrophilic channel (FIG. 2C), asudden diameter shrink of a hydrophobic channel (FIG. 2D), or otherdesigns that are known to person skilled in the art. In the dimensionrange of the microfluidic channel, surface tension of the fluid is moredominant than the inertia force such as gravity.

To simplify the drawings, a design symbol 3001 as shown in FIG. 3 isused to represent the fluidic unit, with a fluidic chamber 3002, aventing port 3003, a microfluidic channel 3004, and a passive valve3005. Two threshold pressures are associated with the passive valve,ΔP_(in) in the direction of fluid entering the chamber and ΔP_(out) inthe direction of fluid leaving the chamber. The threshold pressures canbe of any value, as discussed below:ΔP _(in)>0,ΔP _(out)=0: One-way valve for stopping flow intochamber  [1]ΔP _(in)=0,ΔP _(out)>0: One-way valve for stopping flow out ofchamber  [2]ΔP _(in)>0,ΔP _(out)>0: Two-way valve  [3]ΔP _(in)=0,ΔP _(out)=0: valve provide no pressure barrier  [4]

In operation, two states of the fluidic units should be considered.State 1: the channel 4004 is beneath the fluid (h≥0), as shown in FIG.4A. State 2: the channel 4004 is above the fluid (h<0), as shown in FIG.4B. The parameter h represents the height difference from a fluidsurface 4006 to the microfluidic channel.

When storing fluid in a chamber 4002 without flow, as shown in FIG. 4A(h>=0) and FIG. 4B (h<0), the pressure difference between a venting port4003 (P₀) and the microfluidic channel (P₁) should satisfy that:−ΔP _(in) −ρgh≤P ₀ −P ₁ ≤ΔP _(out) −ρgh, if h≥0  [5]P ₁ −P ₀ ≤ΔP _(in), if h<0  [6]

where ρgh is the hydraulic pressure of the fluid. To transfer fluid intothe chamber, as shown in FIG. 4C (h>=0) and FIG. 4D (h<0), the pressuredifference should satisfy that:P ₁ −P ₀ >ΔP _(in) +ρgh, if h≥0  [7]P ₁ −P ₀ >ΔP _(in), if h<0  [8]

To transfer fluid out of the chamber, as shown in FIG. 4E (h>=0), thepressure difference should satisfy that:P ₀ −P ₁ >ΔP _(out) −ρgh, if h≥0  [9]

No fluid would be transferred out of the chamber in State 1 (h<0), asshown in FIG. 4F, by applying the pressure difference (P₀−P₁). Thisproperty can be utilized as a “self-stop” mechanism, as shown in FIG. 5.The fluid in the chamber is initially above the microfluidic channel5004, as shown in FIG. 5A, and is transferred out of the chamber byapplying the pressure difference (P₀−P₁). When the fluid level falls tothe height of the channel 5004, as shown in FIG. 5B, the fluid transferis stopped automatically, without the need of accurate timing to removethe pressure difference (P₀−P₁). This “self-stop” mechanism helps tosolve the challenge of accurate volumetric control in pneumaticallyactuated cartridges. Nevertheless, the chamber can be tilted until thatchannel 5004 is below the fluidic (from state 2 h<0 into stage 1 h>0) toenable the fluid being further transferred out if needed.

Table 1 summarizes the operations of the fluidic unit.

TABLE 1 Pressure difference (P₀ − P₁) to actuate the fluidic transfer.State No Flow In Flow Out Flow h ≥ 0 −ΔP_(in) − ρgh ≤ P₀ − P₀ − P₁ <ΔP_(out) − ρgh < P₀ − P₁ P₁ ≤ ΔPout − pgh −ΔP_(in) − ρgh h < 0 P₀ − P₁ ≥−ΔP_(in) P₀ − P₁ < No out flow for any −ΔP_(in) (P₀ − P₁)

The pneumatic pressure P₀ applied to the venting port can be adjustedindependently, for example, by an external pressure source such asatmosphere pressure or an internal source such as a pressure controller.The pressure in the microfluidic channel P₁ can be dependent on severalfactors, including the hydraulic pressure propagation along the fluidand air in the channel, flow resistance of the channel, the surfacetension force (fluid versus channel wall interface, fluid versus airinterface, and fluid versus another fluid with different surfacetension, etc.), and pneumatic pressure applied by an external orinternal pressure source, etc. In certain embodiments, the venting portis kept free of fluid. In certain embodiments, the microfluidic channelcan be fully filled of fluid, partially filled of fluid, or free offluid.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control pressures applied to the fluidicunits above, for example, by controlling a pneumatic force applied tothe fluidic chamber via a venting port. In an embodiment, the controlleris configured to control the pneumatic pressure such that In Flow, OutFlow, or No Flow occurs according to the equations above. For example,the controller can control a pneumatic force applied at a venting portto cause P₀ to change to satisfy the above equations and cause the InFlow, Out Flow, or No Flow conditions.

Due to surface tension, the fluid stored in the chamber may have a flattop surface, such as shown in FIG. 4G, or a non-flat top surface, suchas shown in FIG. 4H and FIG. 4I. The fluidic unit works for bothscenarios. Correction can be made by measuring the surface profile tocompensate the fluid volume accuracy. In other embodiments, chemicalssuch as surfactant can be added to the fluidic to modify the surfacetension of the fluid, and thus also change the fluidic surface profile.

FIG. 6 and FIG. 7 show two examples of implementing the fluidic unit.FIG. 6 shows an application of removing bubbles. As illustrated, a fluidcontaining bubbles is transferred into the unit, as shown in FIG. 6A.And after entering the chamber, the bubbles float up and burst as shownin FIG. 6B, due to the floating force introduced by gravity or areplacement force such as centrifugal force. To accelerate the bubbleremoval, a pneumatic vacuum lower than the gas pressure inside thebubbles can be applied to the venting port. The fluidic unit can be usedin a cartridge to remove bubbles in an initial biological sample, forexample, undesirable bubbles in finger-prick blood. The fluidic unit canalso be used to remove bubbles induced in the cartridge, for example,bubbles from thermal cycles of a Polymerase Chain Reaction (PCR).

FIG. 7 shows the application of accelerating fluid mixing. Asillustrated, two fluids are received in the unit, as shown in FIG. 7A.To accelerate mixing, gas (e.g. air) is pumped into fluid to inducechaotic flow, as shown in FIG. 7B. Proper mixing can be quickly achievedwith this operation. Upon the completion of mixing, bubbles can leavethe fluid as shown in the examples of FIG. 6.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control the above fluid mixing.

Design of Fluidic Unit with Variations

The design of the fluidic unit can be modified to have a plurality ofvariations. In an embodiment, the microfluidic channel can be atdifferent positions with respect to the chamber, as shown in FIGS. 8A-C.In another embodiment, the microfluidic channel can be eitherperpendicular or non-perpendicular to the chamber sidewall, as shown inFIG. 8D. In yet another embodiment, the microfluidic channel can have abend, instead of being a straight channel, as shown in FIG. 8E. Inanother embodiment, the fluidic unit can have two or more ofmicrofluidic channels, as shown in FIG. 8F. In another embodiment, therecan be more than one passive valve in one fluidic channel that isaccessing the chamber. The fluidic unit can have a plurality of theabove variations and/or combinations thereof.

For fluid units with more than one microfluidic channel accessing thechamber, the operation of each of the channel can be considered versusthe venting port, such as described in Table 1. Meanwhile, the pressurein each of channel is not fully independent from each other, but rathercoupled by hydraulic pressure of the fluid and air inside the chamberand the channel. For example, as shown in FIG. 8G, two microfluidicchannels 8004 and 8006 are both accessing chamber 8002 and there is nofluid in the chamber. In this scenario, P1 and P2 are coupled byhydraulic pressure of the air inside the chamber and the channel. Whenthere is a pressure difference between P1 and P2, airflow will begenerated between these two channels and balance the pressure differenceagainst the flow resistance of the chamber and the channel.

In another example, as shown in FIG. 8H, two microfluidic channels P1and P2 are both accessing the chamber, and the fluid in the chamber isbelow the height of the channels. In this scenario P1 and P2 are coupledin the same way as in the example of FIG. 8G.

In another example, as shown in FIG. 8I, two microfluidic channels P1and P2 are both accessing the chamber, and the fluid in the chamber isbelow the height of one channel and above the height of the otherchannel. In this scenario, P1 and P2 are coupled by the hydraulicpressure of both the air and the fluid (P1 to chamber by air, P2 tochamber by fluid). When there is pressure difference between P1 and P2,air and/or fluid flow is generated between the two channels, and thepressure difference is balanced again the flow resistance of the fluidand/or air.

In another example, as shown in FIG. 8J, two microfluidic channels P1and P2 are both accessing the chamber, and the fluid in the chamber isbelow the height of both the two channels. In this scenario, P1 and P2are coupled by the hydraulic pressure of the fluid. When there ispressure difference between P1 and P2, fluid flow is generated betweenthe two channels, and the pressure difference is balanced against theflow resistance of the fluid.

As a force such as gravity is pulling the fluid towards the bottom theunit, thus no fluid flow is generated into the venting port. Thus, thepressure difference between the pneumatic pressure applied at theventing port versus the pneumatic pressure in the chamber is balanced byairflow resistance which can be controlled to be relatively minimal.

In fluidic units that have two or more of the microfluidic channels,fluid transfer in each of the channels can be carried out in serial, inparallel, or in a combination of both. For example, FIG. 9 shows afluidic unit that has three microfluidic channels 9004, 9006 and 9008.In FIG. 9A, the fluid transfer in these three channels is carried out inserial. More specifically, in Step 1, a fluid is transferred into theunit via the channel 9004. In Step 2, a fluid is transferred into theunit via the channel 9006. In Step 3, the fluid in the unit istransferred out via the channel 9008. In FIG. 9B, the fluid transfer viathe channel 9004, 9006 and 9008 is a combination of both in serial andin parallel. In Step 1, fluid transfers via the channel 9004 and 9006are carried out in parallel. In Step 2, fluid transfer via the channel9008 is carried out in serial to the previous step.

FIG. 10 shows an example of a fluidic unit 10001 to mix fluids. First,one fluid 10010 is transferred into the unit via a channel 10004, asshown in FIG. 10A, and another fluid 10011 is transferred into the unitvia a channel 10006, as shown in FIG. 10B. The two fluids can be mixedby diffusion, as shown in FIG. 10C, or by the accelerated mixing withbubbles, as shown in FIG. 10D.

FIG. 11 shows an example of using a fluidic unit to transfer fixedvolumes of fluid with the “self-stop” mechanism discussed above. Asshown in FIG. 11A, a fluid volume V₀ can be transferred out a channel11004. The volume V₀ is determined by the initial fluid level and theheight of the channel 11004 with respect to the fluid chamber, and doesnot rely on timing of the pneumatic actuation. As shown in FIG. 11B, afluid volume V₁ can then be transferred out from a channel 11006. Thevolume V₁ is determined by the height difference of the two channels11004 and 11006. In an embodiment, as shown in FIG. 11C, a microfluidicchannel 11008 can be positioned at the bottom of the chamber to transfera fluid volume V₂, which is dependent on the position of channel 11006,to fully drain the unit. In other embodiments, the fluidic unit can havemore of the microfluidic channels to transfer a series of fixed fluidvolumes.

FIG. 12 shows examples for flow relay. FIG. 12A shows a unit for flowrelay with two inlet channels 12004 and 12006 and one outlet channel12008. Fluid flow can be sent from 12004 into the chamber and thendrained out of the chamber by channel 12008. Fluidic flow can also besent from 12006 into the chamber and then drained by channel 12008. Inthis way, two fluid flows (one from 12004 to 12008 and one from 12006 to12008) can be carried out separately and in sequential, but both using12008 as outlet. Similarly, FIG. 12B shows a fluidic unit for flow relaywith one inlet channel 12014 and two outlet channels 12016 and 12018.Fluid flow from channel 12014 to channel 12016 can be carried outseparately and in sequential versus fluid flow from channel 12014 tochannel 12018. FIG. 12C shows a unit for flow relay from two inletchannels 12024 and 12026 to two outlet channels 12028 and 12030. Fluidflow can be carried out separately and in sequential from either one ofthe two inlet channels 12024 and 12026, into either one of the twooutlet channels 12028 and 12030. The operation of flow relay can berepeated more than once in the above units. In other embodiments, therecan be any combination of more than one of the fluid inlet and more thanone of the fluid outlets.

FIG. 13 shows a schematic view for washing particle suspensions influid. Washing is a step frequently used in flow cytometer, moleculardiagnostics and other biological analysis to purified cells, beads orother particles in a fluid sample. FIGS. 13A-D show an exampleembodiment of washing particles with a density higher than fluid. First,a fluid 13001 with particle suspensions is transferred into the chamber,as shown in FIG. 13A, and the particles 13002 are allowed to sediment tothe bottom of the chamber, as shown in FIG. 13B. The excessive fluid isthen drained away via a channel 13016 that is above the particlesedimentations, as shown in FIG. 13C. Afterwards, a wash buffer 13003can be transferred into the chamber to re-suspend the particles, asshown in FIG. 13D. These four steps can be repeated as needed to furtherpurify the particles. For particles 14004 that have lower density thanthe fluid, as shown in FIG. 13E, they are allowed to float up in theunit. The excessive fluid is drained away via a channel 14018 that isbelow the floating particles, on the bottom surface of the chamber. Thesedimentation or floating of the particles can be accelerated bycentrifugation, magnetic field, acoustic waves or other methods that areknown to person skilled in the art.

The fluidic unit can also have other variations. For one example, theunit can be initially supplied with reagents in the chamber, as shown inFIG. 14. The regents can be fluid as shown in FIG. 14A, solid beads asshown in FIG. 14B, and/or a fluid of bead suspensions as shown in FIG.14C. The reagents can also be a dried film coating, as shown in FIG.14D, dried powders as shown in FIG. 14E, dried blocks as shown in FIG.14F, or any other format. As such, the fluid transferred into thefluidic unit can mix and react with the reagents to facilitate furtheranalysis. In an embodiment, the reagents are stored in the chamber priorto the introduction of fluid into the chamber.

In yet other variations, additional features can be added to the unit.FIG. 15A shows the example of a filter membrane 15008 added to thechamber of the unit. Filter membrane 15008 is helpful for the washingprocess, as described in FIG. 13, and also for other process, such asseparating particles from a fluid. By picking a filter with pore sizesmaller than target particles 15001, as shown in FIG. 15B, the filtercan trap the particles and allow fluid to flow through, which is usefulin biological tests, for example, in separating plasma from a wholeblood sample. In other embodiments, filter membrane 15008 can be indifferent orientations, such as in a vertical orientation as shown inFIG. 15C. In yet other embodiments, the fluidic unit can have more thanone filter membrane to capture different target particles (e.g.different size).

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.

Fluidic Circuits with a Plurality of Units

A plurality of the above described fluidic units can be used together toform fluidic circuits for different functions. The fluidic units can beused in serial, in parallel, or in a combination of both, and connectedwith other fluidic circuits. In a preferred embodiment of the fluidiccircuit, any two fluidic units in the circuit are interconnected with nomore than one fluidic conduct. In others embodiments of the fluidiccircuit, there could be more than one fluidic conduct interconnectingtwo fluidic units in the circuit. When a plurality of the units is usedin the circuits, it is useful that the venting ports of the chamber ineach unit are controlled independently. For example, if one venting portis controlled to be connecting with the atmosphere, the pressure in thechamber is then constantly equalized to the atmosphere pressure (orhaving minimal pressure difference). In this way, pressure propagationalong fluid and/or air can be decoupled from unit to unit, whichsimplifies the operation of the fluid circuit.

FIG. 16 shows an example of a fluidic circuit with two fluidic units16011 and 16021 in a serial configuration. A fluid conduit 16101connects channel 16018 of the unit 16011 with channel 16026 of unit16021. FIG. 17A shows an example embodiment of a fluidic circuit withtwo fluidic units 17011 and 17021 in a parallel configuration, which areconnected to a third fluidic unit 17031 in a serial configuration. FIG.17B shows another example embodiment of a fluidic circuit with a firstfluidic unit 17041 in serial to two fluidic units 17051 and 17061 thatare in a parallel configuration. In other embodiments, there can be morefluidic units in the fluidic circuits. FIG. 18 shows one exampleembodiment of a fluidic circuit with six of fluidic units. Giving thedesign of the basic fluidic unit and its operation roles, designing morecomplex circuits can be achieved by a person skilled in the art.

FIG. 19 shows an example embodiment of a fluidic circuit for serialdilution. The function of serial dilution is frequently used inbiological tests such as Complete Blood Count and ELISA assays. Thefluidic circuit has two units 19011 and 19021 in a serial configuration.FIG. 19A shows a fluid sample 19001 transferred into the unit 19011. InFIG. 19B, a first diluent is then transferred into the unit 19011 via asame or separate fluid channel and mixes with the initial sample inchamber 19012 to form a once-diluted sample 19002. Thereafter, a fixedvolume of the once-diluted sample 19002 is transferred into a secondfluidic chamber 19022 of fluidic unit 19021, as shown in FIG. 19C.Finally, a second diluent is transferred into fluidic unit 19021 to mixwith once-diluted sample 19002 and form a twice-diluted sample 19003, asshown in FIG. 19D. In other embodiments, a fixed volume of thetwice-diluted sample 19003 can be transferred into following units forfurther dilution.

T-junctions, which are intersections of fluidic channels, can be usedwith the presently disclosed microfluidic designs. For example, FIG. 20Ashows a T-junction 20104 that is formed by the intersection of threefluidic conduits 20101, 20102 and 20103. However, T-junctions face thecomplexity of pressure balance, so it is preferable to avoidT-junctions. The fluidic unit of the present disclosure can be used toreplace T-junctions, either in cartridges discussed in the presentdisclosure or in other microfluidic cartridges. FIG. 20A shows anexample embodiment of using a fluidic unit 20031 to replace theT-junction 20104 in FIG. 20A. Fluidic unit 20031 acts as a flow relay,as described in FIG. 12, to transfer fluid among the three fluidicconducts 20101, 20102 and 20103 separately. In other embodiments, asshown in FIG. 20B, T-junctions formed by intersections of more thanthree channels can be replaced by the fluidic unit. In yet otherembodiments, multiple T-junctions can be formed in cascade, as shown inFIG. 20C. In an embodiment, fluidic units can be used to replace each ofthe intersections of FIG. 20C.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.

Structure of the Fluidic Units

The fluidic units of the present disclosure can be formed with aplurality of layers. For example, a fluidic unit can be formed with atwo-layer structure, as shown in FIG. 21. FIG. 21A shows the side viewof a basic fluidic unit, and FIG. 21B shows the cross-sectional view ofthe two-layer structure. In this example embodiment, a first layer 21001has cavity structures, and a second layer 21012 is a flat substrate.These two layers bond together to form the fluidic channels and chambersof the unit. The passive valve 21013 of this example is formed with asudden enlargement of the channel geometry and hydrophobic treatment ofthe surface property locally. The venting port 21003 can be a throughhole in the first layer 21001, an opening in the sidewall of the firstlayer 21001, or a through hole in the second layer 21012. The ventingport can access the chamber directly or indirectly via a fluidic channelconnecting to the chamber.

In other embodiments, the first layer 21011 can be formed of materialssuch as thermoplastics (e.g. acrylic, polycarbonate, polyethylene,etc.), silicone, parylene, or other materials such as polymer, plastic,glass, silicon, or other materials known to those skilled in the art offluidics. The cavities of the first layer 21011 can be formed withmanufacturing process such as injection molding, compression embossing,3D printing, CNC, etching, or other process that are known to thoseskilled in the art. In further embodiments, the second layer 21012 canbe a rigid piece or a flexible membrane. The rigid piece can be of samematerial as the first layer or a different material. In an exampleembodiment, the membrane can be a plastic film. In another exampleembodiment, the membrane can be a plastic film laminated with analuminum foil. For embodiments with a membrane as the second layer21012, the membrane can be pierced open during operation of thecartridge. For example, the venting ports can be sealed with themembrane initially and pierced open during operation.

In other embodiments, the second layer 21012 can also have cavities. Asillustrated in FIG. 21C, the fluidic channels and chambers can be formedby any of the following combinations: cavities in the first layer 21011with a flat portion of the second layer 21012, cavities in the secondlayer 21012 with the flat portion of the first layer 21011, and cavitiesin the first layer 21011 with cavities in the second layer 21012. Theterms “first” and “second” are referring to the top and bottom layers inthe drawing and can be used interchangeably. In some embodiments, atleast one layer can be transparent for optical observations andmeasurements.

A fluidic unit according to the present disclosure can also be embodiedin a three-layer structure, as shown in FIG. 22. A middle layer 22003can be added in between the first layer 22001 and the second layer22002. The middle layer 22003 can cover at least a portion part of theinterface between the first and second layers. In some embodiments, themiddle layer 22003 can be a structure layer with cavities, so thatmultiple layers of fluidic channels and chambers can be formed, as shownin FIG. 22A. In another embodiment, the middle layer 22003 can be amembrane, which for example can seal reagents in the fluidic chambersand can be pierced open during operation, as shown in FIG. 22B. Inanother embodiment, the middle layer 22003 can be a metal electrode, thesurface of which can be treated to use as one or more sensors. In otherembodiments, as shown in FIG. 22C, the middle layer 22003 can be a meshstructure to form the filter membrane described in FIG. 15.

Embodiments for Biological Tests: Complete Blood Count

Fluidic circuits including a plurality of the fluidic unit can be usedtogether to form a cartridge for one or more biological tests. Fluidiccircuits can also include other fluidic components to form thecartridges. These components can include but not limit to fluidicchannels, sample retaining chamber, pumps, valves, flow sensors, or anyother component that is known to person skilled in the art. Certainembodiments of the fluidic cartridge can be used for cell analysis inbiological samples, such as a Complete Blood Count (CBC). A CBC analysiscomprises four parts, including analysis of the white blood cells(WBCs), the red blood cells (RBCs), the platelet cells (platelets) andthe hemoglobin.

FIG. 23A shows a fluidic cartridge for a CBC analysis. The fluidiccircuit has four fluidic units 23011, 23021, 23031 and 23041. Thefluidic units 23011 and 23031 are initially loaded with reagentsolutions 23005 and 23006, respectively. To operate the fluidiccircuits, in Step 1, a blood sample is introduced through inlet port23001, drawn into the sample-retaining chamber 23003 by capillary force,and stopped at capillary break 23004. The volume of the blood sample canbe determined based on the geometry of the retaining chamber. In Step 2,valve 23002 is closed to seal off the inlet port 23001. In Step 3, thefirst reagent 23005 is transferred out of the unit 23011, flushing theblood sample in retaining chamber 23003 into fluidic unit 23021 formixing. The volume of the reagent 23005 can be determined with the knownvolume stored in the unit, or with other methods such as the function offixed volume transfer as shown in FIG. 11. The mixing of the bloodsample with the reagent 23005 can be accelerated by pumping air bubbles,and forms a once-diluted sample in fluidic unit 23021. In Step 4, afixed volume of the once-diluted sample is transferred into fluidic unit23041. In Step 5, the second reagent 23006 is transferred into fluidicunit 23041, where it mixes the once-diluted sample and forms atwice-diluted sample. In step 6, a fixed volume of the twice-dilutedsample can be transferred out of the channel 23044 for downstreamanalysis.

In some embodiments, the biological sample can be whole blood, and boththe first reagent 23005 and the second reagent 23006 are isotonicdiluents. In this embodiment, the twice-diluted sample can be used forachieving various dilution ratios of the blood sample, such as dilutionratio of 1:10 to 1:10,000, for the purpose of analysis of WBC, RBCs andplatelets in CBC. The serial dilution of two times is used to achieve ahigh dilution ratio with a lesser diluent volume. In other embodiments,a one-time dilution with one unit can be used. In other embodiments, aserial dilution with more than two units can be used. In anotherembodiment, the first reagent 23005 can be a non-isotonic diluent, andthe second reagent 23006 can be a WBC labeling reagent. In thisembodiment, the once-diluted sample can have RBCs lysed for hemoglobinanalysis. The twice-diluted sample can be used for WBC analysisdownstream.

FIG. 23B shows an example of the above cartridge with a sheathless,microfluidic channel 23007 to perform the cytometer analysis of theWBCs, RBCs and platelets. The inner diameter of the sheathless channel23007 should be larger than target cells for analysis, and be smallenough to minimize coincidence error, i.e. the possibility of multiplecells overlapping. When the diluted sample flows through channel 23007,individual cells can be measured by methods such as optical sensing inflow cytometery, impedance sensing or any other measurement methods thatare known to those skilled in the art. After analysis, the residualsample can be transferred into fluidic unit 23051 as a waste reservoir.With this cartridge device, the total count of WBCs, RBCs and platelets,and the cell indices including but not limited to mean corpuscularvolume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean CorpuscularHemoglobin Concentration (MCHC), and WBC properties including but notlimited to WBC differential (e.g. lymphocyte, monocyte, neutrophil,eosinophil and basophil), can be measured from the blood sample.

FIG. 24 shows one embodiment of a cartridge that integrates the wholeCBC panel. This cartridge includes two duplicates of the cartridge inFIG. 23A. Additional features are added to run the two components of theCBC panel: first, the WBC and hemoglobin analysis, and second, the RBCand Platelet analysis, in one cartridge. One feature is a common inletport 24011 to draw a blood sample simultaneously into two retainingchambers 24014 and 24015, which are used for the RBC/Platelet analysisand WBC/Hemoglobin analysis, respectively. Another feature is a fluidicunit 24009 as flow relay to direct the twice-diluted samples into onesheathless channel 24022 for the cytometer analysis separately. In otherembodiments, additional CBC parameters can also be measured, such asReticulocyte count, Nucleated RBC count, Platelet aggregates, etc. bymodifying the reagents stored in the cartridge.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.For example, the controller can be configured to control the pressureP10 at the venting port of fluidic unit 23011, the pressure P20 at theventing port of fluidic unit 23021, the pressure P30 at the venting portof fluidic unit 23031, the pressure P40 at the venting port of fluidicunit 23041, the pressure P10 at the venting port of fluidic unit 24001,the pressure P20 at the venting port of fluidic unit 24002, the pressureP30 at the venting port of fluidic unit 24003, the pressure P40 at theventing port of fluidic unit 24004, the pressure P50 at the venting portof fluidic unit 24005, the pressure P60 at the venting port of fluidicunit 24006, the pressure P70 at the venting port of fluidic unit 24007,the pressure P80 at the venting port of fluidic unit 24008, the pressureP90 at the venting port of fluidic unit 24009, and/or the pressure P100at the venting port of fluidic unit 24010. The controller can also beconfigured to control pumps and/or valves in any of the fluidicconduits, for example, to allow a pressurized, gravity or capillaryaction flow through the conduits. The pressures can be controlled, forexample, in accordance with the equations described above.

Embodiments for Biological Tests: Flow Cytometer Analysis

FIG. 25 shows a cartridge device for flow cytometer analysis. Thiscartridge has four fluidic units for sample preparation and a sheathlesschannel 25011 for the cytometer measurement. In Step 1, a biologicalsample is drawn by capillary force into retaining chamber 25009 andstops at capillary break 25010. In Step 2, valve 25008 is closed to sealoff inlet 25007. In Step 3, a first reagent 25005 is transferred out offluidic unit 25001 and flushes the sample into the unit 25003 formixing. After being incubated for a certain period of time, the mixtureforms a once-diluted sample. In Step 4, a second reagent 25006 is alsotransferred from fluidic unit 25002 into fluidic unit 25003 for mixingand incubation. After being incubated for a certain period of time, themixture forms a twice-diluted sample, which then flows throughsheathless channel 25011 for sensing such as optical measurements inflow cytometers. The measurement waste is transferred to fluidic unit25004 for storage. In one embodiment, the biological sample is wholeblood, the first reagent is fluorophore-conjugated antibody to labellymphocyte subsets and the second reagent is a lyse solution to break upthe undesired RBCs in the sample. This embodiment can be used forcytometer analysis of lymphocyte subsets that is commonly used inimmunology and infectious diseases diagnostics. In other embodiments,this cartridge device can be used for other cytometer analyses withdifferent samples and reagents. For example, it can be used for ananalysis of bead-based assays, or other cytometer assays known to thoseskilled in the art.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.For example, the controller can be configured to control the pressureP10 at the venting port of fluidic unit 25001, the pressure P20 at theventing port of fluidic unit 25002, the pressure P30 at the venting portof fluidic unit 25003, and/or the pressure P40 at the venting port offluidic unit 25004. The controller can also be configure to controlpumps and/or valves in any of the fluidic conduits, for example, toallow a pressurized, gravity or capillary action flow through theconduits. The pressures can be controlled, for example, in accordancewith the equations described above.

FIG. 26 shows another example embodiment of a cartridge device for flowcytometer analysis. The first four steps are similar to the cartridge ofFIG. 25, collecting a biological sample and forming a twice-dilutedmixture in the unit 26003. In Step 5, the mixture is transferred tofluidic unit 26005. It is noted that fluidic unit 26005 has a filtermembrane 26011 that has a pore size smaller than the target cells of thesample. In Step 6, excessive fluid in the sample is transferred to awaste reservoir unit 26006 and the target cells are collected above thefilter membrane 26011. In Step 7, a third reagent 26010 is transferredinto fluidic unit 26005 to re-suspend the target cells in fluid. Steps 6and 7 together consist of a wash step common in flow cytometer analysisand can be repeated multiple times to purify the target cells from otherundesired components of the sample. In Step 8, the fluid suspension ofthe target cells flows through the sheathless channel 26012 for sensingsuch as an optical measurement. In an embodiment, the biological sampleis whole blood, the first reagent is fluorophore-conjugated antibody tolabel WBC subsets, the second reagent is a non-isotonic solution to lysethe RBCs and the third reagent is an isotonic dilution buffer. Thefilter membrane 26011 can have a pore size smaller than the WBCs butlarger than the RBCs, wherein target WBCs are labeled with thefluorophore-conjugated antibody and purified from other cellularcomponents such as debris of RBC lysis before the cytometer sensing. Inother embodiments, the cartridge device can be used for other cytometeranalysis with different samples and reagents. For example, the cartridgedevice can be used for analysis of bead-based assays, or other cytometerassays known to people skilled in the art. Other cartridge variationscan also be used for flow cytometer analysis.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.For example, the controller can be configured to control the pressureP10 at the venting port of fluidic unit 26001, the pressure P20 at theventing port of fluidic unit 26002, the pressure P30 at the venting portof fluidic unit 26003, the pressure P40 at the venting port of fluidicunit 26004, the pressure P50 at the venting port of fluidic unit 26005,the pressure P60 at the venting port of fluidic unit 26006, and/or thepressure P70 at the venting port of fluidic unit 26007. The controllercan also be configure to control pumps and/or valves in any of thefluidic conduits, for example, to allow a pressurized, gravity orcapillary action flow through the conduits. The pressures can becontrolled, for example, in accordance with the equations describedabove.

Embodiments for Biological Tests: Clinical Chemistry

FIG. 27 shows a cartridge device for clinical chemistry analysis. InStep 1, a biological sample is loaded into the sample-retaining chamber27013 via the inlet 27011. In Step 2, the valve 27012 is closed to sealoff the inlet. In Step 3, a first reagent 27009 is transferred out ofthe fluidic unit 27001 and flushes the sample into fluidic unit 27002for mixing. In Step 4, the mixed sample is transferred into fluidic unit27003 with a filter membrane 27010. This filter stops particles largerthan the pore size and allows other fluidic component to pass through.In Step 5, a known volume of the fluid that passes through the filter istransferred into the unit 27004. In step 6, a plurality of known volumesof the fluid is transferred into a plurality of reaction chambers, e.g.fluidic units 27005, 27006, 27007 and 27008, respectively. The knownvolumes can be determined by the height of the fluid channels withrespect to fluidic unit 27004. In the reaction chambers, the fluid mixesand reacts with reagents 27015 initially stored in the chambersrespectively for sensing measurement. In an embodiment, the biologicalsample can be whole blood, plasma or serum. The first reagent can be anisotonic buffer. The filter membrane can have a pore size small enoughto remove all cellular components such as WBCs, RBCs and platelets fromthe sample. The reagents initially stored in the reaction chambers canbe dried reagents that dissolve when mixed with the fluid, wherein theserum or plasma component of the sample can be diluted, filtered andtransferred to the reaction chambers to react with the reagent beads.Optical measurements such as spectrometer or other measurements can beperformed in each chamber to determine concentrations of a targetclinical chemistry analyte. With a known volume of the biological sampleand a known volume of the isotonic buffer, the initial concentrations ofthe analyte can be calculated from the measurement data. Furthermore,with a known volume percentage of plasma or serum in a whole bloodsample, the initial concentrations of the analyte in terms of plasma orserum can also be obtained.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.For example, the controller can be configured to control the pressureP10 at the venting port of fluidic unit 27001, the pressure P20 at theventing port of fluidic unit 27002, the pressure P30 at the venting portof fluidic unit 27003, the pressure P40 at the venting port of fluidicunit 27004, the pressure P50 at the venting port of fluidic unit 27005,the pressure P60 at the venting port of fluidic unit 27006, the pressureP70 at the venting port of fluidic unit 27007, and/or the pressure P80at the venting port of fluidic unit 27008. The controller can also beconfigure to control pumps and/or valves in any of the fluidic conduits,for example, to allow a pressurized, gravity or capillary action flowthrough the conduits. The pressures can be controlled, for example, inaccordance with the equations described above.

FIG. 28 shows another embodiment of a cartridge device for the clinicalchemistry analysis. In this device, a two-stage filtering can be used toimprove the filter efficiency and to minimize RBC hemolysis. Thefiltering step in FIG. 27 can be replaced with two fluidic units, 28003and 28004, each of which has a filter membrane 28015 and 28016,respectively. In one embodiment, filter membrane 28015 has a pore sizesmaller than RBCs but larger than platelets, whereas filter membrane28016 has a pore size smaller than platelets. In this device, the flowpressure required to drive fluid to pass through membrane 28015 is lowerthan in the design unit 27003 of FIG. 27, so the RBCs can be removedfrom the sample with reduced possibility of hemolysis. The flow pressurerequired to drive fluid to pass through the membrane 28016 is also lowerthan in the design unit 27003 of FIG. 27, so the possibility of breakingup platelets is also reduced. By avoiding hemolysis, the sensitivity andaccuracy of the clinical chemistry analysis can be improved.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.For example, the pressure P10 at the venting port of fluidic unit 28001,the pressure P20 at the venting port of fluidic unit 28002, the pressureP30 at the venting port of fluidic unit 28003, the pressure P40 at theventing port of fluidic unit 28004, the pressure P50 at the venting portof fluidic unit 28005, the pressure P60 at the venting port of fluidicunit 28006, the pressure P70 at the venting port of fluidic unit 28007,the pressure P80 at the venting port of fluidic unit 28008, and/or thepressure P90 at the venting port of fluidic unit 28009. The controllercan also be configure to control pumps and/or valves in any of thefluidic conduits, for example, to allow a pressurized, gravity orcapillary action flow through the conduits. The pressures can becontrolled, for example, in accordance with the equations describedabove.

Other variations of cartridge device can also be used for clinicalchemistry analysis. In different embodiments, various chemistryparameters such as the basic metabolic panels, the complete metabolicpanels, the lipid panels, glucose concentration, C-Reactive Proteinconcentration, HbA1C hemoglobin concentration, D-dimer, Creatinine,Albumin etc. can be measured as well. Various samples such as wholeblood, plasma, serum, urine, etc. can also be measured.

Embodiments for Biological Tests: Immunoassay

FIG. 29 shows a cartridge device for immunoassay tests such as an ELISAassay. In this cartridge, fluidic unit 29001 is a flow relay to transfersample and reagents in sequential to sensing zones. In Step 1, abiological sample 29014 is loaded into a volumetric chamber 29008 via asample inlet 29006. In Step 2, valve 29007 is closed to seal off theinlet port. In Step 3, air is pumped out of the unit 29001 and pushesthe sample 29014 to flow over the sensing zones 29010, 29011, 29012 and29013. In Step 4, a first reagent 29015, a second reagent 29016 and athird reagent 29017 are transferred in serial into and then out of theunit 29001, so as to flow over the sensing zones in sequential. Thesample and reagents enters the waste reservoir unit 29005 after flowingover the sensing zones. In one embodiment, the biological sample can bewhole blood, plasma or serum. The sensing zones can be initially coatedwith a primary antigen and a blocking reagent. The blocking reagent,e.g. a neutral protein such as BSA, blocks sites on the sensing zonesthat are not occupied by the primary antigen. When the sample flowsover, the coated antigen captures the target antibodies onto the sensingzones. The first reagent can be a wash buffer to remove residual sample,and the second reagent can be an enzyme-conjugated secondary antibodythat further binds to the target antibodies captured. The third reagentcan be a substrate to react with the enzyme for colorimetricmeasurement. The sensing zones can be washed with the first reagentmultiple times if needed. In other embodiments, the second reagent canbe a fluorophore-conjugated secondary antibody that allows fluorescentmeasurement without the colorimetric reaction of the third reagent. Inyet other embodiments, the cartridge device can be used with othercombination of reagents for different ELISA assays, such as a direct, anindirect sandwich assay, etc., which are known to person skilled in theart.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.For example, the controller can be configured to control the pressureP10 at the venting port of fluidic unit 29001, the pressure P20 at theventing port of fluidic unit 29002, the pressure P30 at the venting portof fluidic unit 29003, the pressure P40 at the venting port of fluidicunit 29004, and/or the pressure P50 at the venting port of fluidic unit29005. The controller can also be configure to control pumps and/orvalves in any of the fluidic conduits, for example, to allow apressurized, gravity or capillary action flow through the conduits. Thepressures can be controlled, for example, in accordance with theequations described above. The controller can also be configured tocontrol the sensing and analysis that occurs at zones 29010, 29011,29012 and 29013.

FIG. 30 is another embodiment of a cartridge device for immunoassayssuch as an ELISA assay. In comparison to FIG. 29, more units are used tostore reagents. In addition, two or more of the stored reagents can betransferred into fluidic unit 30001 for mixing and reaction, beforebeing further transferred to flow over the sensing zones. In oneembodiment, the reagents 30007, 30008, 30009 and/or 30010 can be mixedin the unit 30001 for chemical or biological reactions to forms afreshly prepared reagent, which can flow over the sensing zones within apredetermined time period. In an embodiment, this cartridge device canbe used to perform an immunoassay for human IgG in blood, which usessilver enhancement for signal amplification. The reagents 30007, 30009can be a solution of silver salts and the reagents 30008, 30010 can be asolution of hydroquinone. These two reagents produce signalamplification upon mixing and are stored in separated units before test.In addition of the devices of FIG. 29 and FIG. 30, other variations ofcartridges can also be designed to perform different immunoassays.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.For example, the controller can be configured to control the pressureP10 at the venting port of fluidic unit 30001, the pressure P20 at theventing port of fluidic unit 30002, the pressure P30 at the venting portof fluidic unit 30003, the pressure P40 at the venting port of fluidicunit 30004, the pressure P50 at the venting port of fluidic unit 30005,and/or the pressure P60 at the venting port of fluidic unit 30006. Thecontroller can also be configure to control pumps and/or valves in anyof the fluidic conduits, for example, to allow a pressurized, gravity orcapillary action flow through the conduits. The pressures can becontrolled, for example, in accordance with the equations describedabove. The controller can also be configured to control the sensing andanalysis that occurs at zones 30017, 30018, 30019, 30020.

Embodiments for Biological Tests: Molecular Diagnostics

Embodiments of this fluidic unit can also be used for moleculediagnostics. FIG. 31 shows an embodiment of a cartridge device to purifynucleic acid from a biological sample. In Step 1, a biological samplewith cells for nucleic acid purification is transferred into fluidicunit 31001 via inlet channel 31010. In Step 2, a first reagent 31013, asecond reagent 31014, a third reagent 31015 and a fourth reagent 31016can be transferred into the unit 31001 in serial for mixing andincubation. In an embodiment, the first reagent 31013 can be aproteinase K solution, which breaks down cell membranes and releasescellular mass and the nucleic acids into the fluid. The second reagent31014 can be a detergent solution that lyses cells and solubilizes thecellular mass excluding the nucleic acids, the third reagent 31015 canbe a binding solution that increases the affinity of the nucleic acidsbinding to a silica surface, and the fourth reagent 31016 can be fluidsuspension of beads that has a silica surface coating. Upon incubation,the beads can capture the released nucleic acids in the fluid at the endof the Step 2. In Step 3, the sample mixture can be transferred intofluidic unit 31006 with a filter membrane 31012. In an embodiment, thepore size of this filter membrane 31012 can be smaller than the beads,and thus trap the beads above the membrane and allow excess fluid topass through. In Step 4, the excess fluid can then be transferred intowaste reservoir unit 31009. In Step 5, a fifth reagent 31017 fromfluidic unit 31007 can be transferred into fluidic unit 31006. In anembodiment, the fifth reagent 31017 can be a wash buffer to purify thecaptured beads. Excess fluid can then be transferred into the wastereservoir unit 31009 and the wash step can be repeated multiple times ifneeded. In Step 6, a sixth reagent 30108 from fluidic unit 31008 can betransferred into fluidic unit 31006. In an embodiment, the sixth reagent30108 can be an elution buffer, which releases the nucleic acids fromthe beads binding. Finally, the elusion buffer containing the releasednucleic acids can be transferred out of channel 31011 for furtheranalysis, whereas the beads are trapped above the filter membrane. Otherembodiments of this cartridge can also involve different reagents,thermal treatment to accelerate or stabilize the reactions, and othervariations to optimize the nucleic acid purification process.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.For example, the pressure P10 at the venting port of fluidic unit 31001,the pressure P20 at the venting port of fluidic unit 31002, the pressureP30 at the venting port of fluidic unit 31003, the pressure P40 at theventing port of fluidic unit 31004, the pressure P50 at the venting portof fluidic unit 31005, the pressure P60 at the venting port of fluidicunit 31006, the pressure P70 at the venting port of fluidic unit 31007,the pressure P80 at the venting port of fluidic unit 31008, and/or thepressure P90 at the venting port of fluidic unit 31009. The controllercan also be configure to control pumps and/or valves in any of thefluidic conduits, for example, to allow a pressurized, gravity orcapillary action flow through the conduits. The pressures can becontrolled, for example, in accordance with the equations describedabove.

Other variations of cartridge devices can further include PCR steps toamplify the purified nucleic acids and/or measurement steps to determinethe quantity of the nuclide acids. FIG. 32 shows a cartridge device forbead-based flow cytometer analysis to determine the quantity of nucleicacids. In Step 1, a sample containing DNA segments is drawn into aretaining chamber 32015. In Step 2, valve 32014 is closed to seal offthe inlet 33005. In Step 3, a first reagent 32008 from fluidic unit32001 can be transferred out of fluidic unit 32001 and flush the sampleinto fluidic unit 32003 for mixing and incubation. In an embodiment, thefirst reagent 32008 can be a fluid suspension of beads that areinitially coated with probes to capture DNA segments. The beads can becoated with multiple types of probes for analysis of multiple types ofDNA segments. In Step 4, a second reagent 32009 from fluidic unit 32002can be transferred into fluidic unit 32003 for mixing and incubationwith the sample. In an embodiment, the second reagent 32009 can be afluorophore-conjugated probe to bind to the captured DNA segments. Uponincubation, the DNA segments captured on the beads are further labeledwith fluorophore. In Step 5, excess fluid can be transferred out offluidic unit 32003 and into a waste reservoir unit 32005, whereas thebeads are trapped above filter membrane 32011. Next, a third reagent32010 can be transferred from fluidic unit 32004 into fluidic unit 32003to re-suspend the beads. In one embodiment, the third reagent 32010 canbe a wash buffer and Step 5 washes away excessive fluorophore to reducethe background noise. This wash step can be repeated multiple times ifneeded. In Step 6, the prepared sample is transferred into sheathlesschannel 32012 for cytometer analysis such as fluorescence detection todetermine the quantity of the DNA segments. The measurement waste istransferred into waste reservoir unit 32006.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.For example, the controller can be configured to control the pressureP10 at the venting port of fluidic unit 32001, the pressure P20 at theventing port of fluidic unit 32002, the pressure P30 at the venting portof fluidic unit 32003, the pressure P40 at the venting port of fluidicunit 32004, the pressure P50 at the venting port of fluidic unit 32005,and/or the pressure P60 at the venting port of fluidic unit 32006. Thecontroller can also be configure to control pumps and/or valves in anyof the fluidic conduits, for example, to allow a pressurized, gravity orcapillary action flow through the conduits. The pressures can becontrolled, for example, in accordance with the equations describedabove. The controller can also be configured to control the sensing andanalysis that occurs at sheathless channel 32012.

Embodiments for Biological Tests: Blood Gas

FIG. 33 shows a cartridge device for blood gas analysis. In Step 1, abiological sample 33008 such as whole blood or plasma or serum is drawinto the retaining chamber 33007 by capillary force and stops at thecapillary break 33009. In Step 2, valve 33006 is closed to seal offinlet 33005, and in Step 3, a first reagent 33010 is relayed fromfluidic unit 33002 to fluidic unit 33003 and into the sensing zones33011, 33012, 33013 and 33013 for measurement. In an embodiment, firstreagent 33010 is a calibration solution. In Step 4, air or a secondreagent is transferred out of the unit 33001 and pushes the blood sample33008 to the chamber for the fluid unit 33003. The blood sample or themixture of the blood and the second reagent is then further transferredto the sensing zones for measurement. The measurement wastes arecollected in reservoir unit 33004. In one embodiment, the sensing zonesare fluidic chambers with exposed electrodes, which can be initiallycoated with reagents for electrochemical sensing of blood gascomponents. In other embodiments, the electrode can be a layer of metalor a pre-manufactured sensor piece.

In an embodiment, a device containing a fluidic cartridge with the abovefluidic units or a device containing the above fluidic units can includea controller configured to control fluid flow through the above fluidicunits, for example, by controlling a pneumatic force applied to afluidic chamber via a venting port or by controlling pumps an/or valvesin fluid communication with microfluidic channels of the fluidic units.For example, the controller can be configured to control the pressureP10 at the venting port of fluidic unit 33001, the pressure P20 at theventing port of fluidic unit 33002, the pressure P30 at the venting portof fluidic unit 33003, and/or the pressure P40 at the venting port offluidic unit 33004. The controller can also be configure to controlpumps and/or valves in any of the fluidic conduits, for example, toallow a pressurized, gravity or capillary action flow through theconduits. The pressures can be controlled, for example, in accordancewith the equations described above. The controller can also beconfigured to control the sensing and analysis that occurs at zones33011, 33012, 33013, 33014.

In addition to the abovementioned embodiments, the cartridges can beused to measure one or multiple of the above biological tests in onecartridge, and/or to perform other biological tests.

Methodology to Compensate Tilting

For embodiments of fluidic units and cartridges that utilize gravity, itis desirable to maintain a vertical position. For example, the accuracyof transferring a fixed volume of fluid is sensitive to tilting awayfrom the vertical position, as illustrated in FIG. 34A. Fluidic unit34001 is designed to transfer a fixed volume V1 with the “self-stop”mechanism, for which the height difference between the two channels34002 and 34003 is h. When the unit is in a tilted position, as shown inFIG. 34B, the height difference between the two channel decreases toh′=h cos θ. The volume transferred V1′ differs form the designed volumewith a deviation of ΔV=V1′−V1, which is dependent on the tile angle θand the geometry of the chamber. Three methodologies are taught in thepresent disclosure to compensate this deviation.

The first methodology is to design a chamber with a desirable geometryto compensate the deviation. For example, the chamber of unit 34001 canbe a cylinder, as shown in FIG. 34C, wherein the diameter d of the fluidstored in the cylinder becomes d′=d/cos θ after tilting, as shown inFIG. 34D. Therefore, the volume deviation can be calculated as follow:ΔV=V1′−V1=V1÷cos θ−V1=(1/cos θ−1)V1  [10]

For another example, the chamber of unit 34001 is a rectangular cuboidas shown in FIG. 34E. When the tilting is only in the direction of theedge L, as shown in FIG. 34F, the length of the cuboid becomes L′=L/cosθ and the width remains w′=w (no tilt in the direction of the edge w).Therefore, the volume deviation can be calculated as following:ΔV=V1′−V1=h cos θ×L/cos θ×w−hLw=0  [11]

The rectangular cuboid geometry is more preferable to the cylindergeometry to compensate the volume deviation, when the tilt is at certainangle. Other variations of the chamber geometry, such as a circular conefrustum, can be used to compensate the volume deviation, when the tiltcan be at any angle.

The second methodology is to measure the tilt angle θ with a tiltsensor, such as a tiltmeter or an inclinometer, wherein the volumedeviation can be calculated, as shown in the examples of Equation [10]and Equation [11]. The calculated volume deviation can be used as aparameter to compensate tilt for the biological test data measured onthe cartridge. Furthermore, tilting of the cartridge can be monitoredbefore, during and after the biological tests continuously. Therefore,not only tilting but also vibration, which can be interpreted ascontinuous changing of tilt angle, can be monitored and compensated aswell. Various tiltmeters or inclinometers can be used for this purpose,either embodied on the cartridge or installed separately in a readerinstrument that receives the cartridge. In one embodiment, the tiltmetercan be a MEMS accelerator-based tiltmeter, which has the merits of lowcost, high reliability, large measurement range, and a resolution of 0.1to 1 degree.

The third methodology is to add a flow sensor 34005 at the outlet of theunit, as shown in the example of FIG. 34G. The flow sensor can measurethe precise volume V1′ being transferred and is independent on thetilting. Various flow sensors can be used for this purpose.

Reader Devices to Work with Cartridges

The cartridges of the present disclosure are inserted into a readerinstrument for signal readout. The reader instrument can be designed toaccept one cartridge at a time, or multiple cartridges at a time. Byrunning multiple cartridges in serial, in parallel, or in a streamlineconfiguration, a high test-throughput can be achieved. The streamlineconfiguration means that multiple cartridges are run in parallel for thesample preparation stage and run in serial for the signal sensing stage.In this streamline configuration, only one set of external sensingcomponents is needed for the signal readout. In other embodiments, thereader instrument can be designed to accommodate only one type ofcartridge, such as cartridge for measuring Complete Blood Count, or toaccommodate multiple types of cartridges, such as cartridges forComplete Blood Count, Blood Chemistry, Immunoassay, etc. In otherembodiments, the reader instrument is designed to read cartridge thatintegrates multiple types of biological tests.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present subjectmatter and without diminishing its intended advantages. It is thereforeintended that such changes and modifications be covered by the appendedclaims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present disclosure. At the very least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the disclosure areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The terms “a” and “an” and “the” and similar referents used in thecontext of the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

Preferred embodiments of the disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects those of ordinary skill in the art toemploy such variations as appropriate, and the inventors intend for thedisclosure to be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the disclosure so claimed areinherently or expressly described and enabled herein.

Further, it is to be understood that the embodiments of the disclosuredisclosed herein are illustrative of the principles of the presentdisclosure. Other modifications that may be employed are within thescope of the disclosure. Thus, by way of example, but not of limitation,alternative configurations of the present disclosure may be utilized inaccordance with the teachings herein. Accordingly, the presentdisclosure is not limited to that precisely as shown and described.

The invention is claimed as follows:
 1. A method for analyzingbiological samples, comprising: receiving a fluid sample into acartridge device, which comprises: a fluidic chamber; at least onemicrofluidic channel in fluid communication with the fluidic chamber;and a venting port configured to apply a pneumatic force to the fluidicchamber; inserting the cartridge device into a reader device to performmeasurements, wherein the cartridge device is positioned in a verticalor tilted position such that at least a portion of the fluid sampleinside the fluidic chamber is pulled by gravity in a direction away fromthe venting port or towards the bottom of the fluidic chamber; andreceiving at least two fluid samples in the fluidic chamber andgenerating air bubbles in the mixture of the two fluid samples toaccelerate mixing.
 2. The method of claim 1, wherein the cartridgedevice comprises at least one valve located within the at least onemicrofluidic channel, the at least one valve configured to allow or stopfluid flow through the at least one microfluidic channel.
 3. The methodof claim 2, wherein the valve is a passive valve configured to allow orstop fluid flow though the microfluidic channel based on a pressuredifference.
 4. The method of claim 1, which includes applying apneumatic force to the fluidic chamber using a controller.
 5. The methodof claim 1, which includes applying a pneumatic force to the ventingport via ambient pressure or atmosphere pressure.
 6. The method of claim1, which includes transferring at least a portion of the fluid sampleout of the fluidic chamber through the at least one microfluidicchannel.
 7. The method of claim 1, which includes transferring at leasta portion of the fluid sample out of the fluidic chamber through a firstmicrofluidic channel and at least another portion of the fluid samplethrough a second microfluidic channel.
 8. The method of claim 1, whichincludes receiving two or more fluid samples sequentially, andtransferring the two or more fluid samples out of the fluidic chamber byflowing through the at least one microfluidic channel sequentially. 9.The method of claim 1, wherein the fluid sample includes air bubbles,and which includes removing at least a portion of the air bubbles fromthe fluid sample.
 10. The method of claim 9, which includes removing atleast a portion of the air bubbles from the sample by gravity.
 11. Themethod of claim 9, wherein at least a portion of the air bubbles in thefluid sample is generated by applying a pneumatic force to the cartridgedevice.
 12. The method of claim 1, wherein the cartridge device furthercomprises a filter membrane with pores inside the fluidic chamber. 13.The method of claim 12, wherein the fluid sample includes particles, andwhich includes separating at least a portion of the particles from therest of the fluid sample by the filter membrane.
 14. The method of claim1, wherein the cartridge device further comprises at least one fluidicstructure for sample measurements, and which includes measuring at leastone sensing signal from the fluid sample in the fluidic structure. 15.The method of claim 1, wherein the fluidic chamber has a volume largerthan the volume of the fluid sample received therein and wherein an airgap exists between the venting port and the fluid sample received in thefluidic chamber.
 16. The method of claim 1, wherein the fluidic chamberhas a size about 0.1 mm to 50 mm in width, 0.1 mm to 50 mm in depth and0.1 mm to 100 mm in height, and wherein the at least one microfluidicchannel has a cross section size about 0.1 μm to 5 mm in width and 0.1μm to 5 mm in depth.
 17. A method for analyzing biological samples,comprising: receiving a fluid sample into a cartridge device, whichcomprises: a fluidic chamber; at least one microfluidic channel in fluidcommunication with the fluidic chamber; and a venting port configured toapply a pneumatic force to the fluidic chamber; and inserting thecartridge device into a reader device to perform measurements, whereinthe cartridge device is positioned in a vertical or tilted position suchthat at least a portion of the fluid sample inside the fluidic chamberis pulled by gravity in a direction away from the venting port ortowards the bottom of the fluidic chamber, wherein: the cartridge devicecomprises at least one valve located within the at least onemicrofluidic channel, the at least one valve configured to allow or stopfluid flow through the at least one microfluidic channel; and the atleast one valve is a passive valve configured to allow or stop fluidflow though the microfluidic channel based on a pressure difference.